Preparation method and application of purified hand-foot-mouth disease virus CVA4

Abstract

The application discloses a preparation method and application of purified hand-foot-mouth disease virus CVA4 and belongs to the technical field of virus purification. The preparation method of the purified hand-foot-mouth disease virus CVA4 comprises the following steps: after a virus harvest liquid is filtered and concentrated, purification is carried out; the purification comprises the following steps: the Capto core400 and an anion filler Capto Q are used in sequence for purification. The hand-foot-mouth disease virus is purified by using the purification process of the Capto core400 combined with the anion filler Capto Q, and the comprehensive effects of high antigen recovery rate, high product purity, large sample loading amount, high antigen content and low protein residue can be achieved.

Description

Technical Field

[0001] This invention belongs to the field of virus purification technology, and in particular relates to a method for preparing and applying purified hand-foot-mouth disease virus CVA4. Background Technology

[0002] Hand, foot, and mouth disease (HFMD) is a globally impactful disease. Currently, multiple HFMD viruses co-circulate, and effective cross-protection is difficult to achieve after immunization. Therefore, developing broad-spectrum or multivalent vaccines that simultaneously target multiple HFMD viruses is crucial. Purification of HFMD virus particles is a critical step in vaccine development and production. HFMD virus particles have an icosahedral structure and are approximately 30 nm in size. Previously, Sepharose 6FF molecular sieves were primarily used for HFMD virus purification. This method yields high-purity virus particles, but it involves significant dilution of the target product during purification, necessitating further concentration. Furthermore, the inherent properties of molecular sieve gels limit the loading volume to no more than 3% of the gel column volume. These characteristics make the process complex, with limited loading volume, and difficult to scale up when using Sepharose 6FF for HFMD virus purification during production. Another study used a purification scheme combining Capto Core 700 with anion exchange packing material, but this system is not suitable for hand-foot-mouth disease virus CVA4. During the purification process, CVA4 enters the Capto Core 700 packing material and is easily affected by hydrophobicity, ion adsorption and other effects, causing the virus to be adsorbed inside the packing material and unable to be discharged, ultimately resulting in poor product recovery rate and purity.

[0003] Therefore, it is urgent to develop new purification methods that can ensure the recovery rate and purity of hand-foot-mouth disease virus CVA4 while also meeting the needs of large-scale production. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for preparing purified hand-foot-mouth disease virus CVA4, which is simple to operate, has high yield and purity, low protein residue, and is suitable for large-scale production.

[0005] Another object of the present invention is to provide the application of the preparation method described above in the preparation of purified hand-foot-mouth disease virus CVA16, CVA10, CVA6, poliovirus, rhinovirus or EV-D68.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing purified hand-foot-mouth disease virus CVA4, comprising: filtering and concentrating the virus harvested fluid, and then purifying it; the purification includes sequential purification using Capto Core 400 and anion exchange filler Capto Q.

[0007] Preferably, the virus harvest fluid is obtained by culturing and collecting cells infected with hand-foot-mouth disease virus CVA4.

[0008] Preferably, the cells comprise KMB17 cells.

[0009] Preferably, the pore size of the filter is 0.6~0.7μm.

[0010] Preferably, the concentration includes tangential flow concentration using a membrane with a molecular weight cutoff of 30-100 kD to concentrate the filtrate to 1 / 80 to 1 / 100 of its original volume.

[0011] Preferably, the purification method of the Capto core400 includes: using 1~5mM PBS as the mobile phase, loading volume of 0.1~1CV, purification flow rate of 1~2cm / min, and collecting the breakthrough peak as the target product.

[0012] Preferably, during the Capto Core 400 purification process, OD is used. 280 UV value is the criterion for judgment, when OD 280 Collection begins when the UV value rises to 5 mAu, OD 280 Collection will stop when the UV value drops to 20 mAu.

[0013] Preferably, the purification method of the anion exchange packing material Capto Q includes: loading the product collected after purification of Capto core400 onto the anion exchange packing material Capto Q, with a loading volume of 0.2~0.5 CV, a purification flow rate of 0.5~1.5 cm / min, and collecting the breakthrough peak as the target product.

[0014] Preferably, in the purification process using the anion exchange resin Capto Q, OD 280 UV value is the criterion for judgment, when OD 280 Collection begins when the UV value rises to 1 mAu, OD 280 Collection will stop when the UV value drops to 1 mAu.

[0015] The present invention also provides the application of the preparation method described above in the preparation and purification of hand-foot-mouth disease virus CVA16, CVA10, CVA6, poliovirus, rhinovirus, or EV-D68, comprising: filtration and concentration of the virus harvested fluid, followed by purification; the purification includes sequential purification using Capto Core 400 and anion exchange packing material Capto Q; the virus harvested fluid comprises virus harvested fluids of hand-foot-mouth disease virus CVA16, CVA10, CVA6, poliovirus, rhinovirus, or EV-D68.

[0016] The beneficial effects of this invention are: This invention provides a method for purifying hand-foot-mouth disease virus (HFMD) CVA4. The invention conducts comparative experiments on Capto Core 400 combined with anion exchange resin, Capto Core 700 with a matching process, and a Sepharose 6FF molecular sieve purification scheme. A comparative experiment was also conducted using Capto Core 400 combined with Capto Q against mumps virus SPA strain. Based on the antigen recovery rate, antigen content detection results, and transmission electron microscopy observation of viral morphology, it is confirmed that purification using Capto Core 400 combined with Capto Q anion exchange resin can prepare high-concentration, high-recovery, and high-purity HFMD CVA4. However, this method is not suitable for the purification of mumps virus.

[0017] This invention significantly increases the loading volume, allowing the packing material to achieve a loading volume of 0.1~1 CV. Traditional processes often use Sepharose 6FF molecular sieves for purification, but due to the inherent properties of the molecular sieve material, the loading volume cannot exceed 3% of the column volume, making it difficult to meet the needs of large-scale production. Simultaneously, this invention effectively preserves viral activity, resulting in a high viral titer in the product, eliminating the need for an additional concentration step after purification. In contrast, the Sepharose 6FF molecular sieve purification process significantly dilutes the target product, requiring subsequent concentration, increasing the operational and production steps. Regarding antigen recovery, the Capto Core 400 combined with the anion exchange packing material Capto Q achieves an antigen recovery rate of 38.18%, significantly higher than the 15.6% recovery rate of the Sepharose 6FF molecular sieve process; while related purification schemes using Capto Core 700 show poor performance in both antigen recovery and product purity. It is evident that the purification process using Capto Core 400 combined with the anionic filler Capto Q of this invention can achieve a comprehensive effect of high antigen recovery rate, high product purity, large sample loading capacity, high antigen content, and low protein residue for the purification of hand-foot-mouth virus, making it suitable for large-scale production. Attached Figure Description

[0018] Figure 1The following are peak diagrams for the purification of hand-foot-mouth disease virus CVA4 using the Capto Core 400 process. A shows the peak diagram of CVA4 virus purified by Capto Core 400; the breakthrough peak was collected as sample #1, and the elution peak with 1M NaCl was collected as sample #2. B shows the peak diagram of sample #1 purified by Capto Core 400 followed by Capto DEAE purification; the breakthrough peak was collected as sample #1, and the elution peak with 1M NaCl was collected as sample #2. C shows the peak diagram of sample #1 purified by Capto Core 400 followed by Capto Q purification; the breakthrough peak was collected as sample #1, and the elution peak with 1M NaCl was collected as sample #2. D shows the peak diagram of sample #1 purified by Capto Core 400 followed by Capto Q ImpRes purification; the breakthrough peak was collected as sample #1, and the elution peaks with 1M NaCl were collected as samples #2 and #3. E shows the peak diagram of sample #1 purified by Capto Core 400 followed by Sepharose 6FF purification; the initial peak was collected as sample #1, and samples #2-#4 were collected after the peak value decreased by half.

[0019] Figure 2 The following are peak diagrams for the purification of hand-foot-mouth disease virus CVA4 using the Capto Core 700 process. A shows the peak diagram of CVA4 virus purified by Capto Core 700, with the breakthrough peaks collected from samples 1# and 2#, and the elution peaks from 1M NaCl collected from samples 3# and 4#. B shows the peak diagram of sample 1# purified by Capto Core 700 followed by Capto DEAE purification, with the breakthrough peaks collected from sample 1#, and the elution peaks from 1M NaCl collected from samples 2# and 3#. C shows the peak diagram of sample 1# purified by Capto Core 700 followed by Capto Q purification, with the breakthrough peaks collected from sample 1#, and the elution peaks from 1M NaCl collected from samples 2# and 3#. D shows the peak diagram of sample 1# purified by Capto Core 700 followed by Capto Q ImpRes purification, with the breakthrough peaks collected from sample 1#, and the elution peaks from 1M NaCl collected from samples 2# and 3#.

[0020] Figure 3 The images show peak diagrams for the purification of hand-foot-mouth disease virus CVA4 using the Sepharose 6FF molecular sieve process. A shows the peak diagram for CVA4 virus purification using Sepharose 6FF. Sample #1 is collected when the UV value drops to half of the highest peak. Sample #2 is collected when the UV value drops to the lowest point. Sample #3 is collected when the UV value rises. Samples #4 and #5 are collected sequentially based on the UV value increase. B shows the peak diagram for purification after concentrating and combining samples #1 and #2 collected by Sepharose 6FF using Capto Q. Sample #1 is collected at the breakthrough peak position, and sample #2 is collected at the 1M NaCl elution peak.

[0021] Figure 4 The values ​​represent the viral content in purified CVA4 virus samples. A shows the viral mRNA content in the CVA4 products purified by each purification process using real-time quantitative PCR; B shows the viral titer in the CVA4 products purified by each purification process using cytological experiments. 1-5# correspond to the peaks collected from each purification process.

[0022] Figure 5 To determine the specificity and purity of viral structural proteins in CVA4 virus purified samples. A shows the VP1 protein content of each sample during purification, detected by Western blot. Wells 1-13 are labeled as follows: Marker, concentrated sample, Capto Core 400 1-2#, Capto Core 400+DEAE 1-2#, Capto Core 400+Q 1-2#, Sepharose 6FF 1-5#. B shows the VP2 protein content of each sample during purification, detected by Western blot. Wells 1-13 are labeled as follows: Marker, concentrated sample, Capto Core 400 1-2#, Sepharose 6FF 1-5#, Capto Core 400+DEAE 1-2#, Capto Core 400+Q 1-2#. C represents the VP3 protein content of each sample during purification as determined by Western blot analysis. Wells 1-14 are labeled as follows: Marker, sample before concentration, sample after concentration, Sepharose 6FF 1-5#, Capto Core 400 1-2#, Capto Core 400+DEAE 1-2#, Capto Core 400+Q 1-2#. D represents the protein content and purity of each sample during purification as determined by silver staining. Wells 1-14 are labeled as follows: Marker, sample before concentration, sample after concentration, Sepharose 6FF 1-5#, Capto Core 400 1-2#, Capto Core 400+DEAE 1-2#, Capto Core 400+Q 1-2#. Wells 1-5 correspond to the collected products from each purification process.

[0023] Figure 6To compare the specificity and purity of viral structural proteins in CVA4 virus purification samples during the process. A shows the VP1 protein content of each sample during purification as determined by Western blot analysis. Wells 1-12 are labeled as follows: Marker, Captocore 400 + Q 1#, Captocore 700 1-4#, Captocore 700 + DEAE 1-3#, Captocore 700 + Q 1-3#. B shows the protein content and purity of each sample during purification as determined by silver staining. Wells 1-12 are labeled as follows: Marker, Captocore 400 + Q 1#, Captocore 700 1-4#, Captocore 700 + DEAE 1-3#, Captocore 700 + Q 1-3#. C shows the VP1 protein content of each sample during purification as determined by Western blot analysis. Wells 1-10 are labeled as follows: Marker, Captocore 400 + Q 1#, Captocore 400 + 6FF 1-4#, Sepharose 6FF 1-2#, Sepharose 6FF + Captocore 700 + DEAE 1-2#, Sepharose 6FF + Captocore 700 + DEAE 1-3#, Sepharose 6FF + DEAE 1-3#, Sepharose 6FF + DEAE 1-3#, Sepharose 6FF + DEAE 1-3#, Sepharose 6FF + DEAE 1-3#, Sepharose 70 ... Q1~2#. D represents the protein content and purity of each sample during the silver staining purification process. The sample loading wells 1-10 are, in order: Marker, Captocore 400 + Q 1#, Captocore 400 + 6FF 1~4#, Sepharose 6FF 1~2#, Sepharose 6FF + CaptoQ 1~2#. Wells 1~4# correspond to the collected products from the purification peaks of each process.

[0024] Figure 7 To compare the specificity and purity of structural proteins in CVA4 virus samples purified using Capto Core 400 and Capto Core 700 coupled with Capto Q ImpRes, respectively, in a cross-sectional comparison, Figure A shows the VP1 protein content of each sample during purification as determined by Western blot analysis. Wells 1-8 are respectively: concentrate, Capto Core 400 + Q 1#, Capto Core 400 + Q ImpRes 1-3#, and Capto Core 700 + Q ImpRes 1-3#. Figure B shows the protein content and purity of each sample during purification as determined by silver staining; wells 1-8 are the same as in Figure A.

[0025] Figure 8This table shows the residual protein concentration, viral antigen content, and purification recovery rate of CVA4 virus purified samples. A represents the residual protein concentration in CVA4 virus purified samples using the Lowry method; B represents the antigen content in the CVA4 virus products purified by each process using ELISA; and C represents the CVA4 virus recovery rate calculated based on the antigen content before and after purification. 1-5# correspond to the collected products in the purification peak chromatograms for each process.

[0026] Figure 9 The purity of CVA4 virus purified samples was analyzed by HPLC. A shows the purity of sample #1 collected after concentration of the CVA4 purified by Capto Core 400; B shows the purity of sample #1 collected after the breakthrough peak of Capto Core 400-Capto Q purification by HPLC; C shows the purity of sample #1 collected after the breakthrough peak of Capto Core 400-Capto DEAE purification by HPLC; D shows the purity of sample #1 collected after the breakthrough peak of Capto Core 400-Capto Q ImpRes purification by HPLC; E shows the purity of a mixed sample of samples #2 and #3 after Capto Core 400-Sepharose 6FF purification by HPLC.

[0027] Figure 10 This section presents a comparative analysis of the purity of CVA4 virus purified samples using HPLC. A represents the purity of the CVA4 sample collected after concentration of the Sepharose 6FF purified harvest solution, determined by HPLC. B represents the purity of sample #1 collected from the breakthrough peak after Sepharose 6FF-Capto Q purification, determined by HPLC. C represents the purity of sample #3 collected from the salt elution peak after Capto Core 700-Capto Q purification, determined by HPLC. D represents the purity of sample #3 collected from the salt elution peak after Capto Core 700-Capto DEAE purification, determined by HPLC. E represents the purity of sample #3 collected from the salt elution peak after Capto Core 700-Capto Q ImpRes purification, determined by HPLC.

[0028] Figure 11 The image shows the results of transmission electron microscopy observation of the viral content and morphological structure of CVA4 virus purified by Capto Core 400 process. The scale bar is 200 nm.

[0029] Figure 12 The image shows the results of virus content and morphological structure detection in CVA4 virus purified samples during the transverse comparison process observed by transmission electron microscopy. The scale bar is 200 nm.

[0030] Figure 13The following are peak diagrams of mumps virus SPA strain purified by Capto Core 400 and Capto Core 700 in combination with Capto Q. A is the peak diagram of mumps virus purified by Capto Core 400; the breakthrough peak was collected from sample #1, and the elution peak with 1M NaCl was collected from sample #2. B is the peak diagram of sample #1 purified by Capto Core 400 followed by Capto Q purification; no obvious breakthrough peak appeared, and the position before the conductivity increased after 1M NaCl elution was collected as sample #0 (subsequent detection found no virus content); the 1M NaCl elution peaks were collected from samples #1 and #2. C is the peak diagram of mumps virus purified by Capto Core 700; the breakthrough peak was collected from sample #1, and the elution peak with 1M NaCl was collected from sample #2. D is the peak diagram of sample #1 purified by Capto Core 700 followed by Capto Q purification; no obvious breakthrough peak appeared, and the 1M NaCl elution peaks were collected from samples #1 and #2.

[0031] Figure 14 This section describes the viral content and recovery efficiency in purified mumps virus samples. A represents the detection of viral mRNA content in the products collected after each purification process using real-time quantitative PCR; B represents the detection of viral titer in the products collected after each purification process using cytological experiments; and C represents the calculation of the mumps virus recovery rate for each purification process based on the viral titer before and after purification.

[0032] Figure 15 The specificity and purity of structural proteins in mumps virus samples were determined by combining Capto Core 400 and Capto Core 700 with Capto Q. A shows the mumps virus protein content of each sample during purification using Western blot with mouse immune serum. Wells 1-6 were labeled as follows: Capto Core 400 1#, Capto Core 400+Q 2#, Capto Core 400+Q 1#, Capto Core 700 1#, Capto Core 700+Q 2#, Capto Core 700+Q 1#. B shows the protein content and purity of each sample during purification using silver staining. Wells 1-7 were labeled as follows: concentrate, Capto Core 400 1#, Capto Core 400+Q 2#, Capto Core 400+Q 1#, Capto Core 700 1#, Capto Core 700+Q 2#, Capto Core 700+Q 1#.

[0033] Figure 16The purity of the mumps virus purified samples was analyzed by HPLC. A shows the purity of sample #1, collected after concentration of the mumps virus from the Capto Core 400 purified harvest solution, determined by HPLC; B shows the purity of sample #2, collected from the salt elution peak after Capto Core 400-Capto Q purification, determined by HPLC; C shows the purity of sample #1, collected from the breakthrough peak after Capto Core 700 purification, determined by HPLC; D shows the purity of sample #2, collected from the salt elution peak after Capto Core 700-Capto Q purification, determined by HPLC.

[0034] Figure 17 The image shows the results of transmission electron microscopy observation of viral content and morphological structure in purified mumps virus samples. The scale bar is 200 nm. Detailed Implementation

[0035] This invention provides a method for preparing purified hand-foot-mouth disease virus CVA4, comprising: filtering and concentrating the virus harvested fluid, and then purifying it; the purification includes sequential purification using Capto Core 400 and anion exchange filler Capto Q.

[0036] In this invention, the virus harvest fluid is preferably obtained by culturing and collecting cells infected with hand-foot-mouth disease virus CVA4; in some embodiments, the cells preferably include KMB17 cells. The culture method can be conventionally selected according to actual needs, preferably culturing until the cells change from long spindle-shaped lesions to round shapes, at which point the virus harvest fluid is collected.

[0037] In this invention, the virus harvest fluid is preferably filtered and concentrated before purification. In some embodiments, the pore size of the filter is preferably 0.6~0.7μm, for example 0.65μm. Filtration removes cell debris from the virus harvest fluid without losing virus particles. The concentration preferably includes tangential flow concentration using a membrane with a molecular weight cutoff of 30~100kD, concentrating the filtrate to 1 / 80~1 / 100 of its original volume; the molecular weight cutoff can be selected as 30, 50, or 100kD, preferably concentrated to 1 / 85, 1 / 90, or 1 / 95 of its original volume. After filtration and concentration, cell lysis debris in the virus harvest fluid is removed, and the virus concentration in the harvest fluid is increased.

[0038] In this invention, the purification process includes sequential purification using Capto Core 400 and Capto Q anion exchange resin.

[0039] In some embodiments, the purification method of Capto Core 400 preferably includes: using 1-5 mM PBS as the mobile phase, a loading volume of 0.1-1 CV, a purification flow rate of 1-2 cm / min, and collecting the breakthrough peak as the target product. The concentration of the PBS is preferably 2, 3, or 4 mM; the loading volume is preferably 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 CV; and the purification flow rate is preferably 1.5 cm / min. When collecting the target product, it is preferably in OD0.05. 280 UV value is the criterion for judgment, when OD 280 Collection begins when the UV value rises to 5 mAu, OD 280 Collection will stop when the UV value drops to 20 mAu.

[0040] In some embodiments, the purification method of the anion exchange packing material Capto Q preferably includes: loading the product collected after purification from Captocore 400 onto the anion exchange packing material Capto Q, with a loading volume of 0.2~0.5 CV and a purification flow rate of 0.5~1.5 cm / min, and collecting the breakthrough peak as the target product; the loading volume is preferably 0.3 or 0.4 CV; the purification flow rate is preferably 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3 or 1.4 cm / min. When collecting the target product, it is preferred to use OD... 280 UV value is the criterion for judgment, when OD 280 Collection begins when the UV value rises to 1 mAu, OD 280 Collection will stop when the UV value drops to 1 mAu.

[0041] This invention employs Capto Core 400 in conjunction with the anionic polymeric resin Capto Q for purification, achieving an antigen recovery rate of 38.18%. Specifically, Capto Core 400 alone achieves a 66.68% antigen recovery rate, while Capto Q alone achieves a 64.89% recovery rate for Capto Core 400. Compared to other purification methods, including Capto Core 400 combined with Capto DEAE (23.62%), Capto Core 400 combined with Capto Q ImpRes (9.65%), Capto Core 400 combined with Sepharose 6FF (11.56%), Capto Core 700 combined with Capto Q (0.82%), Capto Core 700 combined with Capto DEAE (1.11%), Capto Core 700 combined with Capto Q ImpRes (1.39%), and Sepharose 6FF combined with Capto Q (5.16%), this invention demonstrates superior antigen retention, significantly improved target product recovery, and better purification results.

[0042] This invention uses Capto Core 400 in conjunction with the anion exchange resin Capto Q for purification. The Capto Core 400 purification yielded a breakthrough peak sample titer of 9.375 CCID. 50 Further Capto Q purification yielded a breakthrough peak titer of 8.75 CCID. 50 The purification process of this invention can maintain a high viral titer in the purified product, eliminating the need for a post-purification concentration step. Furthermore, the CVA4 virus sample purified using this method has a residual protein concentration below 5 μg / mL, indicating low protein residue. Purity comparison results show that the purified product of this invention achieves a purity of 98.30%, significantly higher than Capto Core 400 combined with Capto Q ImpRes (purity 27.48%). In contrast, when Capto Core 700 is combined with Capto DEAE, Capto Q, or Capto Q ImpRes, the target component is concentrated in the salt elution peak, with product purities of only 35.93%, 53.33%, and 37.21%, respectively. In comparison, this invention not only exhibits better viral titer retention but also significantly higher product purity than Capto Core 700 combinations, demonstrating a clear advantage in overall purification performance.

[0043] This invention also provides the application of the aforementioned preparation method in the preparation and purification of hand-foot-mouth disease virus CVA16, CVA10, CVA6, poliovirus, rhinovirus, or EV-D68, comprising: filtration and concentration of the virus harvested fluid, followed by purification; the purification includes sequential purification using Capto Core 400 and anion exchange resin Capto Q; the virus harvested fluid includes virus harvested fluids of hand-foot-mouth disease virus CVA16, CVA10, CVA6, poliovirus, rhinovirus, or EV-D68. The aforementioned hand-foot-mouth diseases are all assembled from structural proteins VP1, VP2, and VP3 to form an icosahedral structure with a particle size of 25-30 nm and highly similar morphological structures. Experimental verification shows that the preparation method of this invention can achieve purification of various hand-foot-mouth diseases such as CVA16, CVA10, and CVA6, with excellent purity and recovery rate of the obtained products. Enteroviruses such as poliovirus, rhinovirus, and EV-D68 also have an icosahedral structure with a particle size of about 30 nm. The preparation method of this invention is also applicable to the purification of such enteroviruses.

[0044] Experimental results show that the purification method of the present invention can achieve efficient purification of CVA series hand-foot-mouth virus, poliovirus, rhinovirus, and EV-D68. However, when this purification process is used to purify mumps virus SPA strain, high purity and high recovery rate of viral products cannot be obtained.

[0045] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0046] Unless otherwise specified, the following embodiments are all conventional methods.

[0047] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0048] Example 1 A method for preparing purified hand-foot-mouth disease virus CVA4, comprising the following steps: KMB17 cells were infected with hand-foot-mouth disease virus (HFMD) CVA4 and cultured to obtain viral harvest fluid. The viral harvest fluid was filtered through a 0.65 μm filter and then concentrated 90-fold by tangential flow using a 100 kDa membrane to obtain a concentrated solution. The concentrated solution was then purified using 2 mM PBS as the mobile phase. First, it was purified using a Capto Core 400 filter with a loading volume of 0.5 CV and a purification flow rate of 1.5 cm / min. The breakthrough peak was collected as the target product. The collected product was then further purified using an anion exchange resin Capto Q with a loading volume of 0.3 CV and a purification flow rate of 1 cm / min. The breakthrough peak was collected as the target product to obtain purified HFMD virus CVA4.

[0049] Example 2 A method for preparing purified hand-foot-mouth disease virus CVA4, comprising the following steps: KMB17 cells were infected with hand-foot-mouth disease virus (HFMD) CVA4 and cultured to obtain viral harvest fluid. The viral harvest fluid was filtered through a 0.65 μm filter and then concentrated 80-fold by tangential flow using a 100 kDa membrane to obtain a concentrated solution. The concentrated solution was then purified using 1 mM PBS as the mobile phase. First, it was purified using a Capto Core 400 filter with a loading volume of 0.1 CV and a purification flow rate of 2 cm / min. The breakthrough peak was collected as the target product. The collected product was then further purified using an anion exchange resin Capto Q with a loading volume of 0.2 CV and a purification flow rate of 1.5 cm / min. The breakthrough peak was collected as the target product to obtain purified HFMD virus CVA4.

[0050] Example 3 A method for preparing purified hand-foot-mouth disease virus CVA4, comprising the following steps: KMB17 cells were infected with hand-foot-mouth disease virus (HFMD) CVA4 and cultured to obtain viral harvest fluid. The viral harvest fluid was filtered through a 0.65 μm filter and then concentrated 100-fold by tangential flow using a 100 kDa membrane to obtain a concentrated solution. The concentrated solution was then purified using 5 mM PBS as the mobile phase. First, it was purified using a Capto Core 400 filter with a loading volume of 1 CV and a purification flow rate of 1 cm / min. The breakthrough peak was collected as the target product. The collected product was then further purified using an anion exchange resin Capto Q with a loading volume of 0.5 CV and a purification flow rate of 0.5 cm / min. The breakthrough peak was collected as the target product to obtain purified HFMD virus CVA4.

[0051] Experimental Example 1 The production of hand-foot-mouth disease virus CVA4 was carried out using KMB17 cells. The virus harvested fluid was filtered and concentrated, and then purified using Capto Core 400 combined with Capto Q, Capto Core 400 combined with Capto DEAE, Capto Core 400 combined with Capto Q ImpRes, Capto Core 400 combined with Sepharose 6FF molecular sieve, Capto Core 700 combined with Capto Q, Capto Core 700 combined with Capto DEAE, Capto Core 700 combined with Capto Q ImpRes, and Sepharose 6FF molecular sieve combined with Capto Q. Different purification schemes were compared horizontally. Each scheme used an equal volume of the same batch of concentrate and 2 mM PBS as the mobile phase. The specific steps are as follows: S1. Preparation of virus harvest fluid: The hand-foot-mouth disease virus CVA4 was isolated from samples of hand-foot-mouth disease patients. After isolation and identification, a seed bank for vaccine production was established, which is a dedicated seed bank strain for vaccine production. The hand-foot-mouth disease virus CVA4 was used to infect KMB17 cells. After culture, the cells changed from long spindle-shaped to round and were collected to obtain the virus harvest fluid.

[0052] S2. Preparation of concentrated virus solution: The virus harvested solution was filtered through a 0.65 μm filter, and then concentrated to 1 / 80 of its original volume using a 100 kD membrane via tangential flow to obtain a concentrated solution. After virus culture, harvesting, filtration, and concentration, a titer of 7.95 × 10⁻⁶ was obtained. 5 CCID 50 A concentrated solution with an antigen content of 20772 U / mL.

[0053] S3, Purification: (1) The concentrate obtained in step S2 was purified using Capto Core 400 at a loading volume of 0.5 CV and a flow rate of 1.5 cm / min. During the Capto Core 400 purification process, at OD... 280 UV value 5 mAu start collecting, OD 280 Collection was stopped when the UV value dropped to 20 mAu. The collected product was denoted as Capto core 400, with the breakthrough peak collected as sample #1 and the 1M NaCl salt elution peak collected as sample #2. Figure 1 (A). Subsequent testing revealed that the high-purity virus was mainly distributed at location #1; (2) Take 1 / 4 volume of the Capto Core 400 purified breakthrough peak product (position 1#) collected in step (1) and purify it using Capto DEAE. The loading volume is 0.3 CV, and the flow rate is 1 cm / min. At OD... 280 UV value 1 mAu to start collecting, OD 280 Collection was stopped when the UV value dropped to 1 mAu. The collected product was denoted as Capto core 400+ DEAE, with the breakthrough peak collected as sample #1 and the 1M NaCl elution peak collected as sample #2. Figure 1 (B). Subsequent testing revealed that the high-purity virus was mainly distributed at location #1; (3) Take 1 / 4 volume of the Capto Core 400 purified breakthrough peak product (position 1#) collected in step (1) and purify it using Capto Q. The loading volume is 0.3 CV, and the flow rate is 1 cm / min. At OD... 280 UV value 1 mAu to start collecting, OD 280Collection was stopped when the UV value dropped to 1 mAu. The collected product was denoted as Capto core 400+Q, with the breakthrough peak collected as sample #1 and the 1M NaCl salt elution peak collected as sample #2. Figure 1 (C). Subsequent testing revealed that the high-purity virus was mainly distributed at location #1; (4) Take 1 / 4 volume of the Capto Core 400 purified breakthrough peak product (position 1#) collected in step (1) and purify it using Capto Q ImpRes. The loading volume is 0.3 CV, and the flow rate is 1 cm / min. At OD... 280 UV value 1 mAu to start collecting, OD 280 Collection was stopped when the UV value dropped to 1 mAu. The collected product was denoted as Capto core 400+Q ImpRes, with the breakthrough peak collected as sample #1 and the 1M NaCl salt elution peak collected as samples #2 and #3. Figure 1 (D). Subsequent testing revealed that the high-purity virus was mainly distributed at location #1; (5) Take 1 / 4 volume of the Capto Core 400 purified breakthrough peak product (position 1#) collected in step (1) and purify it using Sepharose 6FF molecular sieve. The loading volume is 0.03 CV, and the flow rate is 0.56 cm / min. At OD... 280 Collection began when the UV value peaked; this was for sample #1, where the first large OD peak appeared. 280 When the UV value drops to half of its maximum value, switch to the collection tube for sample #2. OD 280 When the UV value drops to its lowest point, switch to the next collection tube, which is sample #3. Collect sample #4 sequentially based on the increase in UV value. The collected product is denoted as Capto core400+6FF. Figure 1 (E). Subsequent testing revealed that the high-purity virus was mainly distributed in positions 2-3. (6) The concentrate obtained in step S2 was purified using Capto Core 700 at a loading volume of 0.5 CV and a flow rate of 1.5 cm / min. Samples 1# and 2# were collected based on the peak shape and position of the breakthrough peak, and samples 3# and 4# were collected based on the 1 M NaCl elution peak. The collected products were denoted as Capto Core 700 (…). Figure 2 (A). Subsequent testing revealed that high-purity virus was distributed at position 1, but the virus loss was significant; (7) Take 1 / 3 volume of the Capto Core 700 purified breakthrough peak product (position 1#) collected in step (6) and purify it using Capto DEAE. The loading volume is 0.3 CV, and the flow rate is 1 cm / min. At OD 280 UV values ​​rose significantly, and collection began; OD... 280Collection ceased when the UV value dropped to the bottom of the peak. The collected product was denoted as Capto core 700+DEAE, with the breakthrough peak collected as sample #1, and the 1M NaCl salt elution peak collected as samples #2 and #3. Figure 2 (B). Subsequent testing revealed that the high-purity virus was mainly distributed at location #3; (8) Take 1 / 3 volume of the Capto Core 700 purified breakthrough peak product (position 1#) collected in step (6) and purify it using Capto Q. The loading volume is 0.3 CV, and the flow rate is 1 cm / min. At OD... 280 UV values ​​rose significantly, and collection began; OD... 280 Collection ceased when the UV value decreased to the bottom of the peak. The collected product was denoted as Capto core 700+Q, with the breakthrough peak collected as sample #1, and the 1M NaCl salt elution peak collected as samples #2 and #3. Figure 2 (C). Subsequent testing revealed that the high-purity virus was mainly distributed at location #3; (9) Take 1 / 3 volume of the Capto Core 700 purified breakthrough peak product (position 1#) collected in step (6) and purify it using Capto Q ImpRes. The loading volume is 0.3 CV, and the flow rate is 1 cm / min. At OD 280 UV values ​​rose significantly, and collection began; OD... 280 Collection ceased when the UV value dropped to the bottom of the peak. The collected product was denoted as Capto core 700+Q ImpRes, with the breakthrough peak collected as sample #1, and the 1M NaCl salt elution peak collected as samples #2 and #3. Figure 2 (D). Subsequent testing revealed that the high-purity virus was mainly distributed at location #3; (10) The concentrated solution obtained in step S2 was purified using Sepharose 6FF molecular sieve at a loading volume of 0.03 CV and a flow rate of 0.56 cm / min. The first large peak OD was observed. 280 Collection began when the UV value dropped to half of its maximum value; this was for sample #1. OD 280 When the UV value drops to its lowest point, switch to the next collection tube, which is sample #2. At OD... 280 When the UV value increases, switch to the next collection tube, which is for sample #3. Based on OD... 280 As the UV value increased, samples #4 and #5 were collected sequentially, and the collected product was denoted as Sepharose 6FF. Figure 3 (A). Subsequent testing revealed that the high-purity virus was mainly distributed in positions 1-2. (11) After concentrating the 1-2# samples collected by the Sepharose 6FF molecular sieve in step (10) by a 5-fold ratio, the samples were purified using Capto Q at a loading volume of 0.3 CV and a flow rate of 1 cm / min. At OD 280 UV values ​​rose significantly, and collection began; OD... 280 Collection ceased when the UV value dropped to the bottom of the peak. The collected product was denoted as Sepharose 6FF+Capto Q, with the breakthrough peak collected as sample #1 and the 1M NaCl elution peak collected as sample #2. Figure 3 (B). Subsequent testing revealed that the high-purity virus was mainly distributed at location #1.

[0054] Figures 1-3 The results showed that the peak value of Capto Core 400 purification was around 105 mAu, the peak value of Capto DEAE purification was around 3.8 mAu, the peak value of Capto Q purification was around 3.9 mAu, the peak value of Capto Q ImpRes purification was around 1.9 mAu, and the UV peak value of Sepharose 6FF purification was around 112 mAu. The breakthrough peak of Capto Core 700 purification was around 155 mAu, followed by a low breakthrough peak and a salt elution peak. The breakthrough peaks of Capto DEAE, Capto Q, and Capto Q ImpRes purification were below 1 mAu, followed by a relatively high salt elution peak. The UV peak value of Sepharose 6FF purification was 52 mAu, and the breakthrough peak of Capto Q purification was nearly 5 mAu.

[0055] The viral content of each purified product was detected. Real-time quantitative PCR was used to detect the viral mRNA content in the CVA4 products purified using the different purification methods described above. Figure 4 (A); The viral titer in the CVA4 purified products collected by various purification methods was detected by cytological experiments. Figure 4 (B) The results showed that the sample titer of the breakthrough peak obtained from Capto Core 400 purification was 9.375 CCID. 50 The sample titer of the salt elution peak was 5 CCID. 50 In subsequent purification, the Capto DEAE purified breakthrough peak titer was 9.375 CCID. 50 The titer of the salt-eluted peak sample was 5.5 CCID. 50 The Capto Q purification breakthrough peak titer was 8.75 CCID. 50 The titer of the salt elution peak sample was 6 CCID. 50 The Capto Q ImpRes purified breakthrough peak sample titer was 5 CCID. 50The salt elution peak sample titers were 7 and 6 CCID. 50 Sepharose 6FF purified the virus, with the highest titer at position 2 being 8 CCID. 50 The results showed that the purified products obtained by combining Capto Core 400 with Capto DEAE or Capto Core 400 with Capto Q had high viral loads. The breakthrough peak titer of the Capto Core 700 purified sample was 6.5 CCID. 50 The sample titer of the salt elution peak was 5.25 CCID. 50 In subsequent purification, the breakthrough peak titers of Capto DEAE, Capto Q, and Capto Q ImpRes were 5.5 CCID. 50 The sample titer for the salt elution peak is 6.25 CCID. 50 Approximately 500 mg / L. Sepharose 6FF purification yielded five purified products (1-5). Analysis of viral RNA, structural proteins, and antigen content revealed that the antigen was mainly distributed in products 1 and 2, with titers of 6.5 and 6.6 CCID, respectively. 50 The titers of products 3-5# are around 5.5 CCID. 50 The viral titer distribution after purification with Capto Q was highest at the breakthrough peak, at 6 CCID. 50 .

[0056] The specificity and purity of viral structural proteins in each purified product were determined. Using VP1, VP2, and VP3 antibodies, the specificity and purity of structural proteins in each purified product were detected by Western blot. Figures 5-7 The samples before concentration were the virus solution filtered through a 0.65 μm membrane in step S2, and the samples after concentration were the corresponding concentrates from step S2. Results showed that virus particles purified by Capto Core 400 combined with Capto DEAE, Capto Core 400 combined with Capto Q, or Capto Core 400 combined with Capto Q ImpRes were mainly distributed in the breakthrough peak sample; virus particles purified by Capto Core 700 combined with Capto DEAE, Capto Q, or Capto Q ImpRes were mainly distributed in the salt elution peak sample; and virus particles purified by Sepharose 6FF were mainly distributed from the point where the first large peak decreased by half until the second peak appeared. Furthermore, silver staining experiments also demonstrated that the breakthrough peak products of Capto Core 400 combined with Capto DEAE or Capto Core 400 combined with Capto Q showed significant purification effects.

[0057] The viral antigen content in each purified product was detected, and the antigen recovery rate of each purification method was calculated. The residual protein content in the CVA4 virus products purified by each method was determined using the Lowry method. Figure 8 (A); The antigen content in CVA4 virus products purified by each purification method was detected using ELISA. Figure 8 (B) The CVA4 virus recovery rate for each purification protocol was calculated based on the antigen content before and after purification. Figure 8 (C). The results showed that the concentration of residual protein in samples with high-purity antigen distribution sites obtained by Capto Core 400 combined with anionic fillers such as Capto Q or Capto DEAE was below 5 μg / mL. The antigen recovery rate of Capto Core 400 was 66.68%, the total antigen recovery rate of Capto Core 400 combined with Capto DEAE was 23.62%, and the antigen recovery rate of Capto Core 400 by Capto DEAE was 40.15%; the antigen recovery rate of Capto Core 400 combined with Capto Q was 38.18%, and the antigen recovery rate of Capto Q by Capto Core 400 was 64.89%; the antigen recovery rate of Capto Core 400 combined with Capto Q ImpRes was 9.65%, and the antigen recovery rate of Capto Core 400 by Capto Q ImpRes was 15.83%; the antigen recovery rate of Capto Core 400 combined with Sepharose 6FF was 11.56%, and the antigen recovery rate of Capto Core 400 by Sepharose 6FF was 17.33%. The antigen recovery rate of Capto Core 700 purification was 1.68%, the total antigen recovery rate of Capto Core 700 combined with Capto DEAE was 1.11%, the antigen recovery rate of Capto Core 700 combined with Capto Q was 0.82%, and the antigen recovery rate of Capto Core 700 combined with Capto Q ImpRes was 1.39%. The total antigen recovery rate of Sepharose 6FF purification was 15.6%, and the antigen recovery rate of Sepharose 6FF combined with Capto Q was 5.16%.

[0058] The purity of each purified product was determined. The purity of each purified product was analyzed using HPLC hydrogel column chromatography. Figures 9-10The results showed that Capto Core 400 combined with Capto DEAE, Capto Core 400 combined with Capto Q, and Capto Core 400 combined with Sepharose 6FF all yielded high-purity target products, with purities of 98.02%, 98.30%, and 100%, respectively. The Capto Core 400 combined with Capto Q ImpRes peak diagram showed a breakthrough peak purity of only 27.48%, possibly due to the low product concentration. Capto Core 700 harvested samples were distributed in the salt elution peaks. The purities of samples obtained with Capto Core 700 combined with Capto DEAE, Capto Core 700 combined with Capto Q, and Capto Core 700 combined with Capto Q ImpRes were 35.93%, 53.33%, and 37.21%, respectively.

[0059] The morphology and structure of the virus in each purified product were observed using transmission electron microscopy. Figures 11-12 The results showed that Capto Core 400 combined with Capto DEAE and Capto Core 400 combined with Capto Q yielded high-purity, high-content, and morphologically uniform hand-foot-mouth disease virus particles, while Sepharose 6FF yielded samples with lower virus concentrations.

[0060] Based on the above results, the combination of Capto Core 400 composite packing material and Capto Q or Capto DEAE anionic packing material yielded the best virus content, purity, and integrity in the purified product. The antigen recovery rate of the Capto DEAE purified product was slightly lower than that of the Capto Q purified product. Furthermore, the Capto DEAE purification process required a significant amount of time and buffer after CIP to restore the packing material pH to approximately 7.4. In conclusion, Capto Core 400 combined with Capto Q anionic packing material was determined to be the optimal purification condition for CVA4 and other hand-foot-mouth disease viruses.

[0061] Experimental Example 2 KMB17 cells were infected with hand-foot-mouth disease virus CVA16, CVA10, and CVA6 respectively, and then cultured to obtain virus harvest fluid. The hand-foot-mouth disease virus CVA16, CVA10, and CVA6 were then purified according to the preparation method in Example 1.

[0062] Experimental results show that the preparation method in Example 1 can purify various hand-foot-mouth viruses such as CVA16, CVA10, and CVA6, and the purity and recovery rate of the obtained products are excellent.

[0063] Experimental Example 3 The mumps virus SPA strain was produced using Vero cells. The virus harvested solution was filtered through a 0.65 μm filter, concentrated in a 300 kDa membrane, and then purified using Capto Core 400 coupled with Capto Q and Capto Core 700 coupled with Capto Q, respectively. The antigen recovery rate and purity of each purification method were measured. Equal volumes of the same batch of concentrate were used in each method, with 2 mM PBS as the mobile phase. The specific steps are as follows: S1. Preparation of virus harvest fluid: The mumps virus used was isolated from samples of patients infected with mumps. After isolation and identification, a seed bank for attenuated vaccine production was established, which is a dedicated seed bank strain for vaccine production. The mumps virus was used to infect Vero cells. After culture, the cells were collected after they changed from long spindle-shaped to round, and the virus harvest fluid was obtained.

[0064] S2. Preparation of concentrated virus solution: The virus harvested solution was filtered through a 0.65 μm filter, and then concentrated to 1 / 50 of its original volume using a 300 kD membrane via tangential flow to obtain a concentrated solution; after virus culture, harvesting, filtration, and concentration, a titer of 5.25 × 10⁻⁶ was obtained. 5 CCID 50 The concentrated liquid.

[0065] S3, Purification: (1) The concentrate obtained in step S2 was purified using Capto Core 400 at a loading volume of 0.5 CV and a flow rate of 1.5 cm / min. During the Capto Core 400 purification process, at OD... 280 UV value 2mAu to start collecting, OD 280 Collection was stopped when the UV value dropped to 5 mAu. The collected product was denoted as Capto core 400, with the breakthrough peak collected as sample #1 and the 1M NaCl elution peak collected as sample #2. Figure 13 (A). Subsequent testing revealed that the virus was mainly distributed at location #1; (2) The Capto Core 400 purified breakthrough peak product (position 1) collected in step (1) was purified using Capto Q, with a loading volume of 0.3 CV and a flow rate of 1 cm / min. At OD... 280 Collection begins at a UV value of 0.5 mAu, OD 280 Collection was stopped when the UV value dropped to 0.5 mAu. The collected product was denoted as Capto core 400+Q. Samples without obvious breakthrough peaks were collected before the 1M NaCl conductivity peak appeared (sample #0). Samples with the 1M NaCl salt elution peak were collected as samples #1-2. Figure 13 (B). Subsequent testing revealed that the virus was mainly distributed at location #2; (3) The concentrate obtained in step S2 was purified using a Capto Core 700 filtration system with a loading volume of 0.5 CV and a flow rate of 1.5 cm / min. At OD... 280 UV value 2mAu to start collecting, OD 280 Collection was stopped when the UV value dropped to 5 mAu. The sample was collected as sample #1 based on the peak shape and position of the breakthrough peak, and the sample was collected as sample #2 based on the 1M NaCl salt elution peak. The collected product was designated Capto core 700. Figure 13 (C) Subsequent testing revealed that the virus was located at position #1; (4) The Capto Core 700 purified breakthrough peak product (position 1) collected in step (3) was purified using Capto Q, with a loading volume of 0.3 CV and a flow rate of 1 cm / min. At OD 280 Collection begins at a UV value of 0.5 mAu, OD 280 Collection was stopped when the UV value dropped to 0.5 mAu. The collected product was denoted as Capto core 700+Q. Samples without obvious breakthrough peaks and those eluted with 1M NaCl were collected as samples #1 and #2. Figure 13 (D). Subsequent testing revealed that the virus was mainly distributed at location #2.

[0066] Figure 13 The results showed that the purification peak of Capto Core 400 was around 45 mAu, while the elution peak of the combined Capto Q purification salt was around 60 mAu. The purification breakthrough peak of Capto Core 700 was around 35 mAu, while the elution peak of the combined Capto Q purification salt was nearly 70 mAu.

[0067] The viral content of each purified product was detected. Real-time quantitative PCR was used to detect the viral mRNA content in the mumps virus products purified using the different purification methods described above. Figure 14 (A); The viral titer of mumps virus was detected by cytological experiments in the purified products collected by various purification methods. Figure 14 (B) The results showed that the sample titer of the Capto Core 400 purified breakthrough peak was 4.125 CCID. 50 The titer of the salt-eluted peak sample was 3.5 CCID. 50 In subsequent purification, the titer of the Capto Q purified salt elution peak was 3.75 CCID. 50 The Capto Core 700 purification yielded a breakthrough peak sample titer of 4.375 CCID. 50 The titer of the salt-eluted peak was 3.375 CCID. 50 In subsequent purification, the titer of the Capto Q purified salt elution peak was 4 CCID. 50The virus recovery rate of each purification protocol was calculated based on the virus titer before and after purification. Figure 14 (C). The results showed that the antigen recovery rate of Captocore 400 purification was 10%, the antigen recovery rate of Captocore 400 combined with Capto Q was 1.69%, and the antigen recovery rate of Capto Q for Captocore 400 was 22.49%. The antigen recovery rate of Captocore 700 purification was 8%, the antigen recovery rate of Captocore 700 combined with Capto Q was 4.12%, and the antigen recovery rate of Capto Q for Captocore 700 was 30.92%.

[0068] The specificity and purity of viral structural proteins in each purified product were determined. Mouse immune serum was used to detect the specificity and purity of structural proteins in each purified product using Western blot. Figure 15 The results showed that the combination of Capto Core 400 and Capto Core 700 with Capto Q purified viral particles were mainly distributed in the salt-eluted sample. Furthermore, silver staining experiments demonstrated that these purification methods could not yield high-purity target products. The purity of each purified product was determined using HPLC hydrogel column chromatography. Figure 16 The results showed that the purity of the products from each purification process was below 20%, and multiple peaks were observed.

[0069] In addition, the morphology and structure of the virus in each purified product were observed using transmission electron microscopy. Figure 17 The results showed that the purified product contained spherical mumps virus particles with spikes, approximately 100 nm in diameter.

[0070] Based on the above results, it is determined that the purity of mumps virus products purified by the composite packing material Capto Core 400 or Capto Core 700 combined with the anionic packing material Capto Q is low and the antigen recovery rate is low. Therefore, neither Capto Core 400 nor Capto Core 700 combined with the anionic packing material Capto Q is suitable for the purification of mumps virus.

[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing purified hand-foot-mouth disease virus CVA4, characterized in that, include: The virus harvested fluid was filtered, concentrated, and then purified; the purification process included sequential purification using Capto Core 400 and Capto Q anion exchange resin.

2. The preparation method according to claim 1, characterized in that, The virus harvest fluid was obtained by culturing and collecting cells infected with hand-foot-mouth disease virus CVA4.

3. The preparation method according to claim 2, characterized in that, The cells include KMB17 cells.

4. The preparation method according to claim 1, characterized in that, The pore size of the filter is 0.6~0.7μm.

5. The preparation method according to claim 1, characterized in that, The concentration includes using a membrane-encapsulated tangential flow concentration with a molecular weight cutoff of 30-100 kD to concentrate the filtrate to 1 / 80 to 1 / 100 of its original volume.

6. The preparation method according to claim 1, characterized in that, The purification method for Capto Core 400 includes: using 1-5 mM PBS as the mobile phase, loading volume of 0.1-1 CV, purification flow rate of 1-2 cm / min, and collecting the breakthrough peak as the target product.

7. The preparation method according to claim 6, characterized in that, During the purification process of Capto Core 400, OD 280 UV value is the criterion for judgment, when OD 280 Collection begins when the UV value rises to 5 mAu, OD 280 Collection will stop when the UV value drops to 20 mAu.

8. The preparation method according to claim 1, characterized in that, The purification method of the anion exchange packing material Capto Q includes: loading the product collected after purification of Capto core400 onto the anion exchange packing material Capto Q, with a loading volume of 0.2~0.5 CV, a purification flow rate of 0.5~1.5 cm / min, and collecting the breakthrough peak as the target product.

9. The preparation method according to claim 8, characterized in that, In the purification process using the anion exchange resin Capto Q, OD 280 UV value is the criterion for judgment, when OD 280 Collection begins when the UV value rises to 1 mAu, OD 280 Collection will stop when the UV value drops to 1 mAu.

10. The application of the preparation method according to any one of claims 1 to 9 in the preparation and purification of hand-foot-mouth disease virus CVA16, CVA10, CVA6, poliovirus, rhinovirus, or EV-D68, characterized in that, include: The virus harvested fluid is filtered, concentrated, and then purified; the purification includes sequential purification using Capto Core 400 and Capto Q anion exchange packing material; the virus harvested fluid includes virus harvested fluids of hand-foot-mouth disease virus CVA16, CVA10, CVA6, poliovirus, rhinovirus, or EV-D68.

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